Correia J J, Welch M K, Williams R C
Arch Biochem Biophys. 1987 Jun;255(2):244-53. doi: 10.1016/0003-9861(87)90391-2.
Phosphocellulose-purified tubulin has been shown to form a characteristic "ladder" of nonmicrotubular aggregates during nondenaturing gel electrophoresis (J. J. Correia and R. C. Williams, Jr. (1985) Arch. Biochem. Biophys. 239, 120-129). In this paper we describe evidence that the intersubunit bonds responsible for formation of these oligomeric particles are disulfides. Two-dimensional nondenaturing-denaturing gel electrophoresis demonstrates that each aggregate zone is composed of alpha- and beta-subunits of tubulin. Omission of beta-mercaptoethanol during the sodium dodecyl sulfate (SDS)-electrophoresis step causes a pattern of aggregates to appear and implicates disulfide linkages in their stabilization. Molecular weights, estimated from mobilities in the second (SDS) dimension of two-dimensional gels, suggest that the aggregates are crosslinked in units of monomers, not heterodimers. Consistent with this conclusion, alpha- or beta-subunits alone (isolated by isoelectric focusing) will form the same ladder of aggregates. The disulfide crosslinking of tubulin is also achievable in solution. It is favored by high concentrations of alcohol, the presence of oxidizing agents, high pH, and high temperature, conditions that denature tubulin and cause rapid noncovalent aggregation or precipitation. When aggregate formation was monitored as a function of time by SDS-gel electrophoresis in the absence of beta-mercaptoethanol and by quantitative sulfhydryl and disulfide titrations, the most effective conditions for the crosslinking reaction included greater than 75% alcohol, excess H2O2, or excess iodine. These results suggest that proximity of a hydrophobic gel matrix, high pH, the presence of oxidizing agents, high protein concentration, tubulin's propensity to aggregate nonspecifically, and the availability of as many as 20 sulfhydryls in alpha beta-tubulin contribute, during nondenaturing gel electrophoresis, to the spontaneous formation of disulfide-crosslinked tubulin aggregates.
磷酸纤维素纯化的微管蛋白在非变性凝胶电泳过程中已显示形成非微管聚集体的特征性“梯状条带”（J. J. 科雷亚和小R. C. 威廉姆斯（1985年）《生物化学与生物物理学报》239卷，120 - 129页）。在本文中，我们描述了证据表明负责形成这些寡聚颗粒的亚基间键是二硫键。二维非变性 - 变性凝胶电泳表明每个聚集体区域由微管蛋白的α和β亚基组成。在十二烷基硫酸钠（SDS）电泳步骤中省略β - 巯基乙醇会导致聚集体模式出现，并暗示二硫键在其稳定化中起作用。从二维凝胶第二维（SDS）的迁移率估计的分子量表明聚集体是以单体而非异二聚体的单位交联的。与该结论一致，单独的α或β亚基（通过等电聚焦分离）将形成相同的聚集体梯状条带。微管蛋白的二硫键交联在溶液中也可实现。它受到高浓度酒精、氧化剂的存在、高pH值和高温的促进，这些条件会使微管蛋白变性并导致快速的非共价聚集或沉淀。当在不存在β - 巯基乙醇的情况下通过SDS - 凝胶电泳以及通过定量巯基和二硫键滴定监测聚集体形成随时间的变化时，交联反应的最有效条件包括大于75%的酒精、过量的H2O2或过量的碘。这些结果表明，在非变性凝胶电泳过程中，疏水凝胶基质的接近度、高pH值、氧化剂的存在、高蛋白浓度、微管蛋白非特异性聚集的倾向以及αβ - 微管蛋白中多达20个巯基的可用性有助于二硫键交联的微管蛋白聚集体的自发形成。
